PROJECT SUMMARY The Golgi apparatus is a central cellular membrane organelle that processes a wide variety of proteins. To best perform its complex functions, Golgi membranes need to form a unique stacked structure. Notably, abnormal Golgi fragmentation has been described in an increasing number of diseases that affect millions of Americans and countless more worldwide, including cancer and neurodegenerative diseases. Despite this, how the Golgi forms this stacked structure under physiological conditions and how it becomes defective in diseases remain largely unknown. Over the last few years, we have developed a multidisciplinary approach employing biochemistry, cell biology, proteomics and glycomics, in combination with a novel in vitro reconstitution assay, to address these fundamental questions. We found that the Golgi stacking proteins GRASP55 and GRASP65 both form trans-oligomers to ?glue? the Golgi cisternae into stacks. Using GRASPs as tools to manipulate Golgi stack formation, we provided the first evidence that Golgi stacking impedes protein trafficking to ensure accurate glycosylation and sorting. During cell division, the Golgi undergoes a disassembly and reassembly process, which is regulated by phosphorylation that controls cisternal stacking through GRASPs and by monoubiquitination that regulates p97/p47-mediated post-mitotic Golgi membrane fusion. We identified HACE1, syntaxin 5, and VCIP135 as the ubiquitin ligase, substrate, and deubiquitinase, respectively, in the latter process. In Alzheimer's disease (AD), we found that beta-amyloid (A?) accumulation activates Cdk5, which phosphorylates GRASP65 and causes Golgi fragmentation. Significantly, rescue of Golgi structure by expressing a phosphorylation deficient mutant of GRASP65 reduces A? secretion by elevating non-amyloidogenic cleavage of the amyloid precursor protein (APP), implicating the Golgi as a potential therapeutic target for AD treatment. Our overall hypothesis is that Golgi matrix proteins, including GRASPs, organize Golgi membranes into a stacked structure to ensure the fidelity of protein modification, processing, and sorting. This MIRA proposal consolidates funded research on two central questions in cell biology concerning Golgi structure and function: 1) how the stacked Golgi structure is formed, and 2) why Golgi stack formation is important for its function. We will explore the mechanism of Golgi structure formation by focusing on GRASPs, Golgi matrix and membrane fusion proteins, as well as their regulation in the cell cycle. We will determine the structure-function relationship of the Golgi in mitosis when the Golgi stack is completely disassembled, in GRASP-depleted cells where Golgi cisternae are unstacked, and in cells under stress or disease conditions when the Golgi is fragmented. In the next 5-10 years, we hope to build a testable model of multiple molecules that form and maintain the structure of the Golgi while accommodating a variety of trafficking events under physiological and pathological conditions. Our long-term goal is to develop molecular tools to block Golgi defects in AD patients and to delay the development of the disease.